Fermentation process

ABSTRACT

A fermentation medium containing: (a) a source of metabolizable carbon and energy; (b) a source of inorganic nitrogen; (c) a source of phosphate; (d) at least one metal selected from the group consisting of an alkali metal, an alkaline earth metal, a transition metal, and mixtures thereof; and (e) biotin, substantially free of particulate matter and bacteria. A fatty material may be added to an aqueous suspension containing at least one dicarboxylic acid to modify the rheological characteristics of the suspension.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U.S. applicationSer. No. 09/663,963, filed Sep. 19, 2000 and claims the benefit ofpreviously copending provisional application Ser. No. 60/156,791 filedon Sep. 30, 1999, and copending provisional application Ser. No.60/175,174 filed on Jan. 7, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded, at least in part, under a grant from theDepartment of Commerce, NIST-ATP Cooperative Agreement Number70NANB8H4033. The Government may therefore have certain rights in theinvention.

BACKGROUND OF THE INVENTION

This invention relates to an improved fermentation medium and processfor making an aliphatic polycarboxylic acid using said medium.

Long-chain alpha, omega-dicarboxylic acids, i.e., those having a carbonnumber of 9 or higher, are used as raw materials in the synthesis of avariety of chemical products and polymers.

Diacids with carbon numbers greater than four are currently producedalmost exclusively by nonbiological conversion processes. These types ofchemical processes for the production of diacids have a number oflimitations and disadvantages. Each process is restricted to theproduction of diacids of specific carbon chain lengths, based on thestarting material used. For example, the dodecandioic acid processbegins with butadiene, therefore the products of this reaction processare limited to acids with chain lengths in multiples of four. Inaddition, the processes are based on nonrenewable petrochemicalfeedstocks, and the multireaction conversion process produces unwantedbyproducts which result in yield losses, heavy metal wastes, andnitrogen oxides which must be destroyed in a reduction furnace.

Biological conversion processes for the production of diacids have anumber of potential advantages relative to the existing non-biologicalconversion processes. Primary among these is the use of renewablefeedstocks as starting materials and the ability to produce the diacidwithout the generation of hazardous chemical byproducts whichnecessitate costly waste disposal processes.

Another important advantage achieved by using a biological process isthat such a process can easily be adapted to produce a wide variety ofdiacids using the same biocatalyst and the same equipment. Becausecurrent organic chemical syntheses are suited to the production of onlya single diacid, the synthesis of several different diacids wouldrequire the development of a new synthetic scheme for each diacid. Onthe other hand, a yeast biocatalyst can be used to produce diacids ofvarying lengths using the same equipment, media and protocols merely byproviding a different substrate to the yeast.

U.S. Pat. No. 6,004,784, the entire contents of which is herebyincorporated by reference, discloses a semi-synthetic fermentationmedium which employs corn steep liquor and brewers yeast extract inorder to reduce the cost of conventional fermentation media whichcontain expensive, highly standardized yeast extracts and yeast nitrogenbases.

The problem associated with the use of such inexpensive substitutes ismany fold. They result in a fermentation broth having significant odoremissions when sparged with air. Particulate matter, especially combinedwith high levels of bacteria, contained in corn steep liquor and crudeyeast extracts make them difficult to sterilize and contribute to thebioburden on media sterilization equipment. These substitutes alsocontain many unmetabolizable components that contribute to color andcolor stability problems which need to be attended to using additionalpurification steps with their incumbent product losses. The selection ofthese substitutes, while lowering media cost, add additional processcosts. Consequently, there remains a need for a low-cost,biofermentation medium which provides nutrients to support growth of theyeast biocatalysts permitting high specific productivity ofpolycarboxylic acids, polyols, and polyhydroxy acids.

Although long chain dicarboxylic acids have limited solubility in waterin their undissociated or partial salt form, there are many cases whereit is necessary to handle them in an aqueous medium. Examples includefermentation and recovery processes used to produce dicarboxylic acids,interfacial polymerization reactions, emulsion polymerization reactions,and enzymatic reactions.

Aqueous suspensions of long chain dicarboxylic acids tend to be highlyviscous and are therefore difficult to handle. Major operational issuesin fermentation reactions used to prepare dicarboxylic acids includeoxygen transfer, heat transfer, gas hold-up in the fermentation broth,and broth hold-up in the exiting gas (foaming) each being affected bythe Theological characteristics of the broth. Likewise, in filtrationprocesses used to recover dicarboxylic acids from the fermentationbroth, high viscosity leads to impractical filtration rates. Inpolymerization and enzyme reactions, viscosity effects on mass transferwould be a major issue since it is desirable to have rapid, predictablecontact of reagents.

In DE 29 09 420 A1, Watanabe, et al. describes a method for treatingsuspensions of long-chain dicarboxylic acids in cell-free fermentationbroth to improve filtration rates and purity of recovered diacids. Hedemonstrated that by heating the aqueous dicarboxylic acid suspension atpH 4 or below to above 50° C. for 1 hour or more, he could control theparticle size distribution and particle morphology. He describes that bygrowing the particles to an average particle size of 40-50 μm he couldobtain improved filtration rates, lower filter cake moisture, and higherdicarboxylic acid purity in the filter cake. Unfortunately, this methodhas limited utility for many applications where aqueous suspensions oflong chain dicarboxylic acids are encountered.

Many fermentation and enzyme reactions occur over a broader pH range andbelow 50° C. In many cases, enzyme and microbial activity would bereduced or destroyed under the conditions described by Watanabe, et al.Interfacial and emulsion polymerization reactions involving aqueousdicarboxylic acid suspensions could only be limited to thermally stablereagents. There is therefore a need for alternative methods ofcontrolling rheological properties of aqueous suspensions of long chaindicarboxylic acids.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a fermentationmedium and process for making polycarboxylic acids, polyols, andpolyhydroxy acids using said medium. The fermentation medium contains:

(a) a source of metabolizable carbon and energy;

(b) a source of inorganic nitrogen;

(c) a source of phosphate;

(d) at least one metal selected from the group consisting of alkalimetals, alkaline earth metals, transition metals, and mixtures thereof;and

(e) a source of biotin, substantially free of particulate matter andbacteria.

The present invention is also directed to a process for makingpolycarboxylic acids, polyols and polyhydroxy acids involving:

(a) providing an organism capable of producing a polycarboxylic acid, apolyol or a polyhydroxy acid;

(b) providing a substrate capable of being converted into apolycarboxylic acid, a polyol or a polyhydroxy acid by the organism;

(c) providing a fermentation medium containing:

(i) a source of metabolizable carbon and energy;

(ii) a source of inorganic nitrogen;

(iii) a source of phosphate;

(iv) at least one metal selected from the group consisting of alkalimetals, alkaline earth metals, transition metals, and mixtures thereof;and

(v) a source of biotin, substantially free of particulate matter andbacteria; and

(d) fermenting the organism in the fermentation medium.

The present invention also relates to an aqueous suspension containingat least one dicarboxylic acid which also includes water, and a fattymaterial in an amount effective to cause formation of substantially diskshaped, substantially partially spherical shaped or substantiallyspherical shaped dicarboxylic acid particles.

The present invention also relates to a method for modifying therheological properties of an aqueous suspension containing adicarboxylic acid including adding an effective dicarboxylic acidparticle shape forming amount of fatty material to the suspension tocause formation of shaped dicarboxylic acid particles, wherein the shapeis selected from the group consisting of substantially disk shaped,substantially partially spherical shaped and substantially sphericalshaped.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a graphic depiction of the Brookfield viscosity of a 7%aqueous fermentation broth suspension of dicarboxylic acids using afatty material to modify the rheological properties of the broth.

DESCRIPTION OF THE INVENTION

All numbers expressing quantities of ingredients and/or reactionconditions are to be understood as being modified in all instances bythe term “about”.

Through the judicious selection of alternative nutrients that supportmicrobial growth, the invention provides a medium which allows importantpolyfunctional materials to be produced commercially, in large quantity,using a biological conversion process. The invention provides a low costalternative to prior art media and methods for its use that is easilyprepared and sterilized, has a low degree of odor, provides a lowercontribution of impurities loading in subsequent downstream processes,yet results in the production of product in at least as high a yield asconventional prior art methods. It is surprising that the myriad ofundefined components comprising corn steep liquor and yeast extract canbe replaced by relatively few components without sacrificing the qualityof microbial growth and productivity of polyfunctional compounds.

Thus, according to one aspect of the present invention, there isprovided an economical fermentation medium capable of facilitating thebioconversion of various types of organic substrates. The fermentationmedium contains the following necessary components: (i) a source ofmetabolizable carbon and energy; (ii) a source of inorganic nitrogen;(iii) a source of phosphate; (iv) at least one metal selected from thegroup consisting of alkali metals, alkaline earth metals, and mixturesthereof, and (v) a source of biotin, substantially free of particulatematter and bacteria. These materials alone satisfy the basic nutritionalrequirements for growing the microorganism yet produce biomass ofsufficient quantity and quality to conduct the bioconversion.

Suitable sources of metabolizable carbon and energy include but are notlimited to glucose, fructose, maltose, glycerol, sodium acetate,methanol, short chain alcohols, and mixtures thereof. The preferredsource of metabolizable carbon and energy is glucose, preferably aliquid glucose syrup, for example, 95% dextrose-equivalent syrup, oreven lower dextrose-equivalent syrups. Such materials contain smallamounts of disaccharides, trisaccharides, and polysaccharides which canbe hydrolyzed during the fermentation by the addition of an amylaseenzyme such as amylase, glucoamylase and cellulase. Thus, glucose can beprovided in situ in a reaction simultaneous with the biooxidation.

Inorganic sources of nitrogen include but are not limited to alkalimetal nitrates such as sodium or potassium nitrate, ammonium salts suchas ammonium sulfate, ammonium chloride, ammonium nitrate and ammoniumacetate. A prefered inorganic nitrogen source is ammonia or ammoniumhydroxide.

Another necessary component of the fermentation medium of the presentinvention is a source of phosphate which includes anyphosphate-containing compounds. Examples thereof include, but are notlimited to, potassium phosphate, sodium phosphate, and ammoniumphosphate. A particularly preferred source of phosphate for use in thepresent invention is potassium phosphate.

Suitable metals for use in the fermentation medium include alkalimetals, alkaline earth metals, transition metals, and mixtures thereof.Particularly preferred metals include potassium, calcium, and magnesium,in combination.

The last critical component of the fermentation medium is biotin whichis substantially free of particulate matter and bacteria.

Any of the medium components may be added as a part of the initialsterile media charge, sterilized as concentrated aqueous solutions forlater addition to the fermentation medium, or may be included in largeexcess in the fermentor inoculum for carry-over into the productionfermentor.

In order to avoid problems associated with odor emission, colorinstability, and contamination, it is imperative that the biotin be freeof particulate matter and bacteria which promote such problems andrequire additional process purification steps. The quantity of biotinrequired will be several orders of magnitude smaller than the organicnitrogen sources it replaces in prior art media.

The fermentation medium of the present invention contains in aqueoussolution: (a) from about 10 g/l to about 60 g/l, preferably from about20 g/l to about 40 g/l of a source of metabolizable carbon and energy,preferably glucose; (b) from about 50 ppm to about 2000 ppm, preferablyfrom about 50 ppm to about 250 ppm, and most preferably from about 100ppm to about 250 ppm, of nitrogen provided by the inclusion of aninorganic nitrogen source; (c) from about 1 g/l to about 10 g/l,preferably from about 1 g/l to about 7 g/l, of a source of phosphate,preferably potassium phosphate; (d) from about 0.01 g/l to about 2 g/l,preferably from about 0.01 g/l to about 1 g/l of at least one metalselected from the group consisting of alkali metals, alkaline earthmetals, and mixtures thereof, preferably a mixture of calcium andmagnesium; and (e) from about 1 μg/l to about 2000 μg/l, preferably fromabout 4 μg/l to about 200 μg/l, and most preferably from about 4 μg/l toabout 20 μg/l of biotin, wherein the biotin is substantially free ofdeleterious particulate matter and bacteria.

The water present in the fermentation medium may be a process waterpurified by distillation, deionization, or softening. Preferred sourcesof water include those from a municipal water distribution system, aprocess recycle stream, or well water wherein adjustments in mineralcontent may need to be taken into account for minerals already containedin these sources of water. For example, the water together with otherrequired ingredients may already contain sufficient mineral componentsto provide all or substantially all the required minerals for growth ofthe organism.

The fermentation media of the present invention satisfies the basicnutrient requirements for the growth of the microorganism, whileminimizing the addition of organic and inorganic components that requireseparation from the product for disposal as waste and contribute toprocess odor emissions. In contrast to fermentation media compositionsknown in the art, the media of the present invention contains noparticulate matter, is particularly amenable to continuous sterilizationin an automated medium preparation process, and is inexpensive. Despitethe nutritionally lean media compositions provided by the presentinvention, there is broad flexibility in formulating the media whileachieving high productivities of polycarboxylic acids, polyols, andpolyhydroxy acids.

Various types of auxiliary components may also be employed in thefermentation medium of the present invention in order to further enhancethe biofermentation process. Examples thereof include, but are notlimited to, various types of trace metals, chelating agents,anti-foaming agents, and the like.

The fermentation media of the present invention may be used with anypolycarboxylic acid-producing, polyol-producing and polyhydroxyacid-producing yeast, and a wide variety of substrates. For example, inthe event that the desired product is a polycarboxylic acid, such as adiacid, any type of fatty acid, fatty acid ester, or alkane substratemay be used. Examples of suitable substrates for the production ofdiacids include, but are not limited to, lauric acid, myristic acid,palmitic acid, stearic acid, oleic acid, palmitoleic acid and methylesters thereof, and mixtures thereof. Examples of alkanes includedodecane, dodecene, tridecane, tetradeance, octadecane, and the like.

Thus, according to another aspect of the present invention, there isprovided a process for making a polycarboxylic acid, a polyol, or apolyhydroxy acid. For exemplary purposes only, the process of thepresent invention will be described with reference to makingpolycarboxylic acids, and specifically diacids, as the desired endproduct.

The process may be operated over any pH range where the microorganismcan grow and catalyze the desired conversion reaction. A preferred andespecially advantageous pH range is in the acidic regime, i.e., a pH ofabout 7 or less. While the low pH improvement can be applied to anyfermentation process, it is especially advantageous when applied to afermentation process which produces polycarboxylic acids. Suitable pHcontrol reagents are ammonia, ammonium hydroxide solution, concentratedpotassium or sodium hydroxide.

The organic substrate can be any organic compound that is biooxidizableto a mono- or polycarboxylic acid. Such a compound can be any saturatedor unsaturated aliphatic compound or any carboxylic or heterocyclicaromatic compound having at least one terminal methyl group, a terminalcarboxyl group and/or a terminal functional group which is oxidizable toa carboxyl group by biooxidation. A terminal functional group which is aderivative of a carboxyl group may be present in the substrate moleculeand may be converted to a carboxyl group by a reaction other thanbiooxidation. For example, a lipase enzyme, for example, can be addedduring the fermentation step to liberate free fatty acids from anotherwise non-metabolizable ester.

Alkanes are a type of saturated organic substrate which are useful inpracticing the process according to the invention. The alkanes can belinear or cyclic, branched or straight chain, substituted orunsubstituted. Particularly preferred alkanes are those having fromabout 4 to about 25 carbon atoms, examples of which include but are notlimited to butane, hexane, octane, nonane, dodecane, tridecane,tetradecane, octadecane and the like.

Examples of unsaturated organic substrates which can be used in theprocess according to the invention include but are not limited tointernal olefins such as 2-pentene, 2-hexene, 3-hexene, 9-octadecene andthe like; unsaturated carboxylic acids such as 2-hexenoic acid andesters thereof, oleic acid and esters thereof including a triglycerylesters having a relatively high oleic acid content, erucic acid andesters thereof including triglyceryl esters having a relatively higherucic acid content, ricinoleic acid and esters thereof includingtriglyceryl esters having a relatively high ricinoleic acid content,linoleic acid and esters thereof including triglyceryl esters having arelatively high oleic acid content; unsaturated alcohols such as3-hexen-1-ol, 9-octadecen-1-ol and the like; unsaturated aldehydes suchas 3-hexen-1-al, 9-octadecen-1-al and the like. In addition to theabove, the organic substrate which can be used in the process accordingto the invention include alicyclic compounds having at least oneinternal carbon—carbon double bond and at least one terminal methylgroup, a terminal carboxyl group and/or a terminal functional groupwhich is oxidizable to a carboxyl group by biooxidation. Examples ofsuch compounds include but are not limited to3,6-dimethyl-1,4-cyclohexadiene; 3-methylcyclohexene;3-methyl-1,4-cyclohexadiene and the like.

Examples of the aromatic compounds that can be used in the processaccording to the invention include but are not limited to arenes such aso-, m-, p-xylene; o-, m-, p-methyl benzoic acid; dimethyl pyridine, andthe like. The organic substrate can also contain other functional groupsthat are biooxidizable to carboxyl groups such as an aldehyde or alcoholgroup. The organic substrate can also contain other functional groupsthat are not biooxidizable to carboxyl groups and do not interfere withthe biooxidation such as halogens, ethers, and the like.

A cosubstrate may be provided to the culture during that stage of thefermentation when the culture is actively oxidizing the substrate toproducts. This is especially true if no net useable carbon or energy canbe recovered from the substrate reaction itself, then this carbon andenergy is provided by the cosubstrate.

The cosubstrate is a fermentable carbohydrate such as glucose, fructose,or maltose; or other fermentable organic compound, for example,glycerol, sodium acetate, methanol, or short chain alcohols; or mixturesthereof. The preferred cosubstrate is glucose, preferably a liquidglucose syrup, for example, 95% dextrose-equivalent syrup, or even lowerdextrose-equivalent syrups. Such materials contain small amounts ofdisaccharides, trisaccharides, and polysaccharides which can behydrolyzed during the fermentation by the addition of an amylase enzymesuch as α-amylase, glucoamylase and cellulase. Thus glucose can beprovided in situ in a reaction simultaneous with the biooxidation.

It is convenient, but not absolutely necessary, if the carbon and energysource used to grow the biomass is the same as the cosubstrate used todrive the oxidative conversion reaction. The practical benefit is thatfewer raw materials need be handled and the various stages of thefermentation can be well integrated. Thus a single cosubstratesterilization and delivery system can be used to deliver both the carbonand energy source to grow the biomass and the cosubstrate to drive theoxidation reaction.

The microorganism that can be used in the process according to theinvention is any microorganism capable of biooxidizing the substrate asdefined herein. The microorganism can be any microorganism in which betaoxidation is partially or completely blocked by disruption, inactivationor deletion of one or more acyl CoA oxidase gene(s); any yeast in whichbeta oxidation is partially or completely disrupted by inactivation ordeletion of one or more acyl CoA oxidase gene(s); any Candida strainwhere beta oxidation is partially or completely disrupted byinactivation or deletion of one or more acyl CoA oxidase gene(s). Whenthe fermentation process involves the biooxidation of a substrate to acarboxylic acid, the microorganism will normally be a yeast. Such amicroorganism is capable of oxidizing the substrate in such a way thatthe mono- and/or dicarboxylic acids formed are not further oxidized bydegradation leading to chain shortening. However, the process accordingto the invention pertains to the use of any microorganism.

Yeast strains known to excrete alpha, omega-dicarboxylic acids as aby-product when cultured on alkanes or fatty acids as the carbon sourceare set forth in U.S. Pat. No. 5,254,466, the entire contents of whichare herein incorporated by reference. These strains are partially orcompletely oxidation-blocked strains; that is, they are geneticallymodified so that the chromosomal POX4A, POX4B and both POX5 genes havebeen disrupted. The substrate flow in this strain is redirected to theomega-oxidation pathway as the result of functional inactivation of thecompeting β-oxidation pathway by POX gene disruption. A completelyoxidation-blocked strain is a C. tropicalis strain, strain H5343 (ATCC20962), which is described in U.S. Pat. No. 5,254,466.

Another suitable strain is one in which one or more reductase genes areamplified resulting in an increase in the amount of rate-limitingomega-hydroxylase through P450 gene amplification and an increase in therate of substrate flow through the ω-oxidation pathway. Strains whichselectively increase the amount of enzymes known to be important to theoxidation of fatty acids are also preferred. Such strains containincreased copies of the CYP and CPR genes. These genes have beenidentified as those relating to the production of the ω-hydroxylasecomplex catalyzing the first step in the oxidation pathway. Strain HDC1is an example of a strain that contains multiple copies of the CYP 52A2Agene integrated into the genome of strain H5343. This strain and similarstrains are described in copending application Ser. No. 60/083,798,filed on May 01, 1998, the entire contents of which are incorporatedherein by reference. Other strains that can be used with the methods ofthis invention are C. tropicalis strains HDC1, HDC5, HDC10, HDC15,HDC20, HDC23, HDC 23-1, HDC 23-2, and HDC 23-3 as are described inPCT/US99/20797, the entire content of which is hereby incorporated byreference.

Suitable sources of inorganic nitrogen include any ammonium-containingcompounds. Examples thereof include, but are not limited to, alkalimetal nitrates or ammonium salts such as ammonium sulfate, ammoniumchloride, ammonium nitrate and ammonium acetate. Preferred nitrogensources are ammonium salts or compounds that generate ammonia throughthe metabolic action of the organism like urea. A particularly preferredsource of inorganic nitrogen for use in the present invention isammonia.

The fermentation process can be modified by utilizing a triglyceride fator oil as the source of both the organic substrate and cosubstrate. Alipase, formulated with the fermentation broth, hydrolyzes or splits thefat or oil into fatty acids and glycerine. Glycerine consumption by theorganism serves to drive the splitting reaction to completion whilesupplying the energy necessary to convert the free fatty acids to theircorresponding dibasic acids. Lipases that are oleo-specific areparticularly preferred. Oleo-specific lipases exhibit a high selectivityfor a triglyceride having a high oleic acid content and selectivelycatalyze the hydrolysis of the oleate ester groups. Examples of sucholeo-specific lipases include but are not limited to the lipasesproduced by Pseudomonas sp, Humicola lanuginosa, Candida rugosa,Geotrichum candidum, and Pseudomonas (Burkholderia). A particularlypreferred lipase is UNLipase from Geotrichum candidum ATCC No. 74170described in U.S. Pat. No. 5,470,741, the entire contents of which areincorporated herein by reference.

The fermentation step is preferably carried out in three stages, growthphase, induction phase, and conversion phase. Each phase may be operatedat the same or different fermentor conditions of temperature, pH,aeration, etc. Growth phase begins when the cell culture is introducedinto the fermentor and a rapid phase of growth ensues. Growth may beexponential or sub-exponential depending on the composition of themedium employed. This continues until the culture reaches linear growthas measured by a decrease in oxygen consumption. Linear growth occurswhen growth is limited by the rate of addition of a key nutrient to themedium; thus growth becomes proportional to the rate at which thelimiting nutrient is supplied. Usually, in the process of thisinvention, the key nutrient will be the cosubstrate. The second phase isthe induction phase. In the induction phase key metabolic activities areinitiated that begin the desired conversion of substrate to product. Theculture can be maintained in a linear growth phase for a period of timebefore inducing the culture. The inducing agent will usually be thesubstrate itself, but for compounds that do not initiate their ownconversion, another inducing agent may be used in concert with thesubstrate. The induction phase can be used to transition the rapidgrowth phase to the conversion phase. The fermentation moves into thenext phase, conversion phase, when the substrate is converted toproduct.

During the conversion phase, the fermentation broth is in an acidic pHrange of between 2 and 7. The preferred operating pH range is from about3.5 to 7.0, even more preferred range is from about 5.0 to about 6.5. pHmay be controlled by automatic titration using a strong base, examplesof which are, sodium hydroxide solution, potassium hydroxide solution,ammonium hydroxide solution, or ammonia gas. Note that by operating thefermentation in this pH regime compared to prior art methods thatammonia can be used for pH control since it will react to form the nonvolatile aqueous ammonium ion. Prior art methods for making dicarboxylicacid when operated in the alkaline pH regime would not effectively makethe aqueous ammonium ion thus causing undesirable emissions of ammoniavapor in the offgas and toxic ammonia accumulation in the broth. Thefermentation can be carried out at a temperature of from about 26° C. toabout 40° C.

In the first stage, a culture medium is inoculated with an activeculture of beta-oxidation blocked microorganism such as a beta-oxidationblocked C. tropicalis strain where a period of rapid exponential growthoccurs. The pH of the culture medium is controlled by the addition ofbase, examples include but are not limited to ammonium hydroxide,potassium hydroxide, sodium hydroxide or ammonia gas. The cosubstrateaddition to the fermentor is preferably a fed-batch addition during theconversion phase. The end of exponential growth phase is marked by adepletion of glucose, a rapid increase in dissolved oxygen, and, mostsensitively, by a rapid increase in offgas oxygen and decrease in offgasCO₂. In the absence of an inducing substrate, biomass will continue toaccumulate at a rate proportional to the glucose feed rate (i.e., lineargrowth). It is desirable to initiate the conversion phase either at theend of exponential growth or at a point when the desired biomass levelhas been attained. If the oxygen transfer characteristics of theproduction vessel are such that the desired biomass level can not bereached by maintaining the culture in exponential growth, then acombination of exponential growth followed by a linear glucose-limitedgrowth phase (induction phase) can be used to achieve a given biomasslevel.

To maintain the culture in a healthy state during the transition toglucose limited linear growth, it may be desirable to start a glucosefeed during growth so that glucose will always be available to theculture. The glucose level in the initial medium charge is selectedbased on the total glucose desired for growing the microorganism lessthe amount that will be fed during growth. The glucose feed may bestarted at any time from the beginning of growth. However because thereis some variation in inoculum quality and lag phase after inoculatingthe production vessel, it is preferred to start the glucose feed duringexponential growth phase at some predetermined biomass concentration, asjudged by direct biomass measurements, or indirect methods like opticaldensity, carbon dioxide evolution rate, or oxygen uptake rate. Theglucose may be a corn syrup refined or unrefined. The glucose may be fedfrom the top of the vessel through the vapor space as a continuousstream or as a continuous series of pulses or impulses. The glucose feedmay also be fed into the vessel under the surface especially into aregion of high shear near the agitator to attain a rapid distributionthroughout the vessel. In large vessels there may be multiple glucosefeed points within the same vessel.

The conversion phase where the substrate is oxidized is initiated byadding an inducer and the substrate containing an oxidizable methylgroup. In the case of alkanes, fatty acids, fatty acid methyl esters andfatty acid salts; these substances and combinations thereof induce theirown oxidation to dicarboxylic acids and may be useful for inducing theoxidation of other substances. The substrate may be added batchwise oras a continuous feed or as a series of continuous pulses or impulses.The substrate may be added from the top of the vessel through the vaporspace or under the surface especially into a region of high shear. Inlarge vessels there may be multiple substrate feed points. The oxidationis conducted at an acidic pH less than 7 and preferable near or belowthe pKa of the carboxyl group being formed by the oxidation or ispresent in the substrate.

It is preferable to progressively decrease the glucose feed rate to thefermentor as the conversion proceeds to prevent the accumulation ofbiomass and triacylglycerol esters. With the conversion of commercialoleic acid, the accumulation of intracellular storage vacuoles is auseful indicator of the glucose feed rate needing adjustment.Adjustments are done by decreasing the feed rate by about 5% to 25% andusually about 10% per each 24 hours of the conversion phase. Otherindicators could also be used such as base utilization rate, CO2evolution rate, or respiratory quotient.

One problem often encountered in the fermentation process is theformation of carboxylic acid soaps and foaming as a result of working inthe traditional alkaline pH range. In an alkaline environment,carboxylic acids form soaps which lead to unwanted foaming of thefermentation broth. The result of the soap formation is a decrease inthe pH of the broth which is adjusted by adding a base such as causticto the broth to maintain the pH of the broth at the desired value. Also,addition of glucose to the basic environment results in formation ofcarbonates that affect the pH of the fermentation broth. To compensatefor the effects on the pH of soap and carbonate formation, largequantities of base must be added to maintain the pH of the broth at thedesired level.

It has surprisingly been found that by carrying out the fermentationprocess in an acidic pH range of from 2 to 7, preferably from 3 to 7 andeven more preferably from 5 to 6.5, rather than a caustic pH range,foaming is substantially reduced and the formation of carbonate fromcarbon dioxide produced during the metabolism of glucose is reducedthereby substantially reducing the amount of raw material needed tocontrol the pH of the fermentation broth. By carrying out thefermentation process at an acidic pH, these problems are substantiallyreduced resulting in decreased amounts of base used during thefermentation process. There is a net consumption of cation equivalentsover anion equivalents during the growth reaction that contributes to adepression of culture medium pH. It is desirable to control the mediumpH within the range from 2-7, and preferably from about 3-6.5 duringgrowth by the addition of base on pH control. Among the bases useful forthis purpose are ammonium hydroxide, ammonia, sodium hydroxide, andpotassium hydroxide. It is desirable to select a base composition thatprovides one or more of the major nutrients consumed during growth suchas ammonium hydroxide, ammonia, or potassium hydroxide. The growthmedium formulation is adjusted to take into account the addition of thepH control reagent. Using NH₄OH or ammonia gas to control the pH reducesthe number of raw materials needed to grow the microorganisms bycombining the pH control agent and the nitrogen source into one rawmaterial added to the fermentor.

With respect to using nitrogen sources such as ammonia or ammoniumhydroxide it is necessary to have only 250 ppm or less nitrogen sourcepresent in the medium to initiate growth and the consumption of these pHcontrol reagents. The ammonia concentration will therefore remain nearlyconstant during growth of the culture. Thus the desired concentration ofammonia in the medium at the time of induction can be convenientlypreselected by adding ammonia or ammonium salts to the initial mediumcharge. Useful sources of ammoniacal nitrogen for the initial fermentorcharge include ammonia phosphate, ammonium sulfate, ammonium nitrate,ammonia, urea, and ammonium hydroxide.

Another aspect of the invention relates to a formulation of afermentation medium useful for propagating Candida tropicalis and givesa high productivity for converting substrates having oxidizable methylgroups to carboxylic acids. By adjusting feed rates of materials intothe fermentation broth, the growth of the microorganism can becontrolled. The major nutrients consumed during growth of themicroorganism on glucose are ammoniacal nitrogen, potassium, magnesium,phosphate, and sulfate. Sodium and calcium are not consumed, howevercalcium should be present at a concentration of about 5-50 ppm or moreto obtain normal growth depending on the formulation of the inoculummedium. Trace minerals and biotin are also included in the medium. Thebiotin may be a relatively pure grade or supplied as a more crude gradesuch as in biotin yeast, yeast extracts, or corn steep liquor.

Useful sources of phosphate are ammonium phosphate, potassium phosphatemono, di, and tribasic, sodium phosphate, mono, di, and tribasic, andphosphoric acid. Useful sources of potassium are potassium sulfate,potassium phosphate, mono, di, and tribasic, and caustic potash.

A fermentation aid can be used in the oxidation of alkanes and/or theoxidation of fatty acids. The preferred fermentation aid is a fatty acidester, a particularly preferred fermentation aid is a methyl ester. Onemajor benefit of the use of such a fermentation control agent is in foamcontrol and controlling the fluid characteristics of the fermentationbroth. If product chain length distribution is important in ultimateproduct performance, it is desirable to select a chain length of themethyl ester that resembles the alkane or fatty acid since the methylester will also be converted to product. The ester may be added batchwise or blended into the feed. If blended into the feed, it may compriseabout 10% or less and preferably about 1% or less of the feed. Somecommercial fatty acids naturally contain some methyl esters and may beused directly as substrates for dicarboxylic acid production. Forexample, EMERSOL® 267 (Cognis Corporation) is a commercial oleic acidcontaining about 1% or less methyl esters and has been found to be agood substrate in this invention. A typical technical grade of oleicacid, EMERSOL® 267, that is used in the process has the approximatecomposition 0.3% C12, 2.4% C14, 0.6% C14:1, 4.7% C16, 4.6% C16:1, 0.2%C17, 0.8% C18, 69.9% C18:1, 10.5% C18:2, 0.3% C18:3.

In the case of alkane oxidations to dicarboxylic acids, it has furtherbeen found useful to use a fatty acid or fatty acid salt as afermentation aid. This results in a better distribution of the alkanethroughout the fermentation broth. The fatty acid may be added batchwiseor may be formulated into the alkane feed. If formulated into the feed,fatty acid or fatty acid salt may typically comprise about 10% or lessand preferably about 5% or less of the feed. Here too, if product chainlength distribution is important in ultimate product performance, it isdesirable to select a chain length distribution of the fatty acid thatresembles the alkane since the fatty acid will also be converted toproduct.

It has surprisingly been found that particular combinations of fattymaterials, for example fatty acids and fatty esters, cause fine aqueoussuspensions of long chain dicarboxylic acids to form highly definedparticles at temperatures below 50° C. Other fatty materials might alsobe used separately or in combination, including fatty alcohols, fattyethers, etc. and are considered a part of this invention. The resultingsuspensions have desirable shear-thinning characteristics thatfacilitate their handling and practical application. It is desirablethat the particles so formed in the suspension have sizes within anarrow size distribution. This aspect of the invention is well suitedfor application in large-scale production equipment. Indeed, superiorperformance occurred in such equipment compared with laboratory scaleequipment. This suggests that wall effects may play a role in particlegrowth.

Useful fatty materials include saturated and unsaturated fatty acids C10-C20 and their esters that include, but are not limited to, sunflowerfatty acids, sunflower fatty acid methyl esters, tallow fatty acids, andtallow fatty acid methyl esters. Examples of typical commercialcompositions of these materials are shown in Table 1. Each compositionlisted below is commercially available from Cognis Corp., Cincinnati,Ohio.

TABLE 1 Compositions of Example Additives High oleic sunflowerHigh-oleic fatty acids Sunflower fatty Emery ® Methyl acid methyl MethylAdditive 244 Tallowate esters Oleate Trade Edenor ® Emery ® Edenor ®ME-V Emery ® Names PK 1805 2203 05 2301 Fatty Acid Fatty AcidDistribution (%) C10 0.05 — C12 0.11 0.65 — C14 0.11 2.80 — 3.0 C14:1 —0.47 — 2.0 C15 0.15 0.45 0.29 C15:1 0.21 — C16 3.87 23.82 4.17 4.0 C16:10.09 2.46 0.10 6.0 C17 — 1.26 — 1.0 C17:1 0.68 — C18 4.73 19.00 4.48C18:1 84.45 42.20 87.4 76.0 C18:2 5.24 2.54 2.91 7.0 C18:3 0.43 0.25 —1.0 C20:1 0.30 0.33 —

The dicarboxylic acids prepared in fermentation broths typically existas needle-like crystals from 1-10 μm in length. The composition of thediacids reflect the chain length of the substrate being oxidized. Suchfermentation broth can be highly viscous to the point of being solid.When a fatty material as described herein is applied to such afermentation, it is possible grow, in situ, substantially disk-shaped,substantially partially spherical (e.g., cup-shaped) and/orsubstantially spherical dicarboxylic acid particles about 15-40 μm ormore in diameter and about 1-5 μm in thickness at temperatures belowabout 50° C. In one embodiment of the present invention, substantiallyspherical particles were generated having a diameter of approximately 1cm. Thus, it is contemplated that dicarboxylic acid particles producedin accordance with the present invention may range from about 1 μm toabout 1 cm or more in diameter and/or thickness. When fatty materialsdescribed herein are applied to fermentations generating or containingdicarboxylic acids, the resulting broth has a low apparent viscosityallowing fermentation broth handling to continue under optimalconditions. For convenience, the term “disk shaped” may be used hereinto also refer to substantially partially spherical particles.“Substantially” is used herein to mean both approximately and precisely.

In the case of fermentation reaction generating dicarboxylic acids, adesirable benefit of using such fatty materials is that these reagentadditives can also potentially be converted to dicarboxylic acids. Thissimplifies the recovery of the dicarboxylic acids from the broth so thatadditional processing to remove the additive is not needed.

Certain commercially available fatty acid preparations can be useddirectly to cause formation of disk shaped particles because, in theirmanufacturing process, fatty derivatives are left behind that cause thedesired particle growth and size distribution in the fermentation.Examples of these include Emersol® 267 Oleic acid (commerciallyavailable from Cognis Corp., Cincinnati, Ohio), which contains lowlevels of methyl ester, and partially-hydrolyzed fatty acids, whichcontains low levels mono-, di-, and tri-glycerides. In the case of adicarboxylic acid fermentation, one can therefore make a judiciousselection of substrate from commercially available substrates havingbeen informed of the methods of this invention.

It is contemplated that fatty materials may be used in the variousfermentation media described herein along with a variety of antifoamssuch as those of the silicone and polypropylene glycol types withoutinhibitory effect on particle growth.

The fatty material may be added to the aqueous suspension ofdicarboxylic acids batchwise or fed-batch. In general the levels offatty materials used may range from about 10 ppm to about 5% or more,but preferably about 50 ppm to about 1%, based on the mass ofdicarboxylic acid. Particle growth occurs so long as a minimal level ofadditives is present. It is believed that the fatty acid additives aidthe dicarboxylic acid particle growth while fatty acid esters and otherfatty derivatives aid in providing a narrow distribution of particlesizes.

In the case of a fermentation generating dicarboxylic acids fromhydrocarbons it is desirable to add a combination of fatty derivativesincluding fatty acids and fatty acid methyl esters. It is mostconvenient to blend these materials into the hydrocarbon feed in orderto simplify the preparation, sterilization, and feeding of the additivesto the fermentation.

Likewise, in the case of a fermentation generating dicarboxylic acidsfrom a fatty acid, it is desirable to add a similar corresponding methylester. Thus the additive will not alter the chain length distribution ofthe dicarboxylic acid product.

Many combinations and applications are possible in practicing thisinvention having disclosed these methods for growing dicarboxylic aciddisk particles and describing the benefits on rheological properties ofthe suspension.

It has surprisingly been found that by maintaining dissolved oxygenconcentration levels below about 25% and preferably below 20%, relativeto saturation with air, that there is a reduction in the amount ofcosubstrate needed during the conversion phase. When the concentrationof dissolved oxygen is below these levels, glucose is more efficientlyutilized in providing energy for oxidation. Consequences of adding toomuch glucose include the further accumulation of biomass andtriacylglycerol esters with diminished carboxylic acid production duringthe conversion phase.

Triacylglycerol ester formation, in oleaginous yeasts, can be controlledor minimized in part by adjustment of the magnesium concentration orratio of magnesium to phosphate in the initial medium charge. Thepreferred magnesium concentration is about 0.1 to about 1.0 gm/L andmore preferably about 0.2 to about 0.8 g/L concentration in thefermentation inoculum. The preferred phosphate: magnesium ratio is 20:1to about 2:1, preferably 15:1 to about 3:1.

It has also surprisingly been found that the fermentation broth duringthe conversion phase accumulates a heat-labile catalase-like activitythat consumes H2O2. This may interfere with glucose concentration assaysthat measure H2O2 produced from glucose oxidase.

A preferred embodiment of the process according to the invention is thepreparation of 9-octadecenedioic acid by biooxidation of oleic acid.While any grade of oleic acid can be used as the substrate, a typicaltechnical grade oleic acid consists of the following carboxylic acids:0.42% C₁₂; 2.7% C₁₄; 0.86% C_(14:1); 6.3% C₁₆; 4.6% C_(16:1); 0.93% C₁₇;2.8 C₁₈; 71.8% C_(18:1); 8.3% C_(18:2); 0.58% C_(18:3). The oleic acidcan also be a high oleic acid grade obtained from a fatty oil of aHelianthus annuus (sunflower seed oil) species described, for example,in U.S. Pat. No. 4,627,192, the entire contents of which areincorporated herein by reference. Other high oleic acid varieties ofoilseeds may also be used in this process. Such oils are very rich inoleic acid and contain at least 70-80% by weight of oleic acid.

The present invention will be better understood from the examples whichfollow, all of which are intended for illustrative purposes only, andare not meant to unduly limit the scope of the invention in any way. Inall the examples, component concentrations are shown in their anhydrousforms. Commercially available hydrates of the listed components may beused if the concentration is appropriately adjusted to include waters ofhydration.

EXAMPLE 1

Fermentation media in accordance with the present invention wereprepared, the components of which are listed in Tables 2 and 3, below.

TABLE 2 Synthetic Production Medium Components Concentration (g/L)Glucose 27.0 Ammonium Sulfate 7.0 Potassium Phosphate, Monobasic 5.1Magnesium Sulfate 0.5 Calcium Chloride 0.1 Citric Acid 0.06 FerricChloride 0.023 Biotin 0.0002 Trace Metals: Boric Acid 0.0009 CupricSulfate 0.00007 Potassium Iodide 0.00018 Ferric Chloride 0.00036Manganese Sulfate 0.00072 Sodium Molybdate 0.00036 Zinc Sulfate 0.00072Water Balance SAG 471 ® Antifoam 0.8 ml

The medium components of Table 2 were heat sterilized in a suitablemanner to avoid any precipitation reactions, then combined in a sterilefermentor vessel upon cooling. The complete, uninoculated medium wasfound to be completely clear with a slight straw color and no strongodors. Addition of the antifoam yielded slight turbidity. Candidatropicalis H5343 ALK 2-1 was grown under sterile conditions on themedium listed in Table 2 in a stirred, aerated fermentor with an initialliquid volume of 12 L. The sterile culture medium was inoculated with a5% inoculum of Candida tropicalis H5343 ALK 2-1 and grown at 35° C., pH5.8 for approximately 10 hours with agitation and aeration ratessufficient to keep the dissolved oxygen above 20%. When the culturestopped growing exponentially and the dissolved oxygen began to rise,the conversion phase was commenced by starting a continuous feed streamof Exxon Developmental Fluid 137 (a hydrocarbon containing approximately94.4% Tridecane, the balance being primarily dodecane) mixed with thefermentation aids 1.25% Emersol ®267 (a technical grade of oleic acid)and 1.25% Emery® 2203 (a technical grade of methyl tallowate) at a rateof 0.7 g/L/hr. Simultaneously, the temperature in the fermentor wasreduced from 35° C. to 30° C., the aeration rate was reduced to 0.4 vvm,and 0.4 bar of backpressure was applied to the vessel. The pH wasmaintained between 5.8-5.9 during the growth and conversion phase with6N KOH. A continuous glucose feed stream was started to the fermentor ata rate of 1.58 g/L/hr glucose when the biomass concentration reachedapproximately 10 g/L. The glucose feed rate during conversion wasreduced between 0-15% on a daily basis based upon the microscopicobservation and assessment of accumulating storage vacuoles within theyeast cell. 7 ml of PPG (polypropylene glycol) antifoam was added to thefermentor during conversion to control mild foaming. After 50 hours ofthe conversion phase, the whole broth in the fermentor contained 41.5g/Kg 1,13-tridecanedioic acid.

EXAMPLE 2

TABLE 3 Synthetic Production Medium Components Concentration (g/L)Glucose 27.0 Potassium Phosphate, Monobasic 4.9 Magnesium Sulfate 0.6Calcium Chloride 0.1 Citric Acid 0.06 Ferric Chloride 0.023 Biotin0.000012 Trace Metals: Cupric Sulfate 0.00007 Manganese Sulfate 0.00432Zinc Sulfate 0.00072 Citrate 0.00708 Water Balance SAG 471 ® Antifoam0.6 ml

The medium components of Table 3 were sterilized in a suitable manner toavoid any precipitation reactions and combined in a sterile fermentorvessel. The complete, uninoculated medium was found to be completelyclear with a slight straw color and no strong odors. Candida tropicalisH5343 HDC 23-3 was grown under sterile conditions on the medium listedin Table 3 in a stirred, aerated fermentor with an initial liquid volumeof 12 L. The sterile culture medium was inoculated with a 3% inoculum ofCandida tropicalis H5343 HDC 23-3 and grown at 35° C. for approximately12 hours with agitation and aeration rates sufficient to keep thedissolved oxygen above 20%. The pH was adjusted to and maintainedbetween 5.8-5.9 during the growth phase with the addition of 6N NH₄OHwhich was also the source of inorganic nitrogen in the medium. When theculture stopped growing exponentially and the dissolved oxygen began torise, the conversion phase was commenced by adding an inducing chargeand simultaneously starting a continuous feed stream of High OleicSunflower Fatty Acids (containing 84.4% oleic acid, 5.2% linoleic acid,4.7% stearic acid, 3.9% palmitic acid, with the balance comprising smallamounts of eicosanoic acid (20:0), eicosaenoic acid (20:1),pentadecanoic acid, lauric acid, and myristic acid) at a rate of 2.0g/L/hr. Simultaneously, the temperature in the fermentor was reducedfrom 35° C. to 30° C., the aeration rate was reduced to 0.4 vvm, and thepH control reagent was switched from NH₄OH to NaOH. The pH wasmaintained between 5.8-5.9 during the conversion phase with 6N NaOH. Acontinuous glucose feed stream was started to the fermentor at a rate of1.22 g/Uhr glucose when the biomass concentration reached approximately10 g/L. The glucose feed rate during conversion was reduced between0-45% on a daily basis based upon the microscopic observation andassessment of accumulating storage vacuoles within the yeast cell. Noadditional antifoam was used during the conversion phase. After 50 hoursof the conversion phase, the whole broth in the fermentor contained 71g/Kg total dicarboxylic acids.

EXAMPLE 3

TABLE 4 Synthetic medium formulation Medium Components Concentration(g/L) Glucose 20.0 Ammonium Sulfate 0.5 Potassium Phosphate, Monobasic5.1 Magnesium Sulfate 0.9 NaCl 0.5 Calcium Chloride 0.1 Biotin 0.0002Trace Metals: Boric Acid 0.00075 Cupric Sulfate 0.00006 Potassium Iodide0.00015 Ferric sulfate 0.04 Ferric Chloride 0.0003 Manganese Sulfate0.0006 Sodium Molybdate 0.0003 Zinc Sulfate 0.0006 Water Balance SAG471 ® Antifoam 2 drops

The medium components of Table 4 were sterilized in a suitable manner toavoid any precipitation reactions, then combined in a sterile fermentorvessel. The medium was found to be completely clear with a slight colorand little odor. Addition of the antifoam yielded slight turbidity. ThepH of the medium was initially adjusted to pH 5.8 using 6N ammoniumhydroxide solution. Candida tropicalis H5343 (ATCC 20962) was grown onthis medium in a stirred and aerated fermentor using a 4% inoculumprepared in a medium having similar composition. Exponential growthbegan following a brief lag phase. A glucose feed was started at a rateof 1.25 g/L/hr based on the initial medium volume when the culturecontained about 5 g/L biomass dry weight as judged by optical densitymeasurements. Exponential growth continued for about nine hours when theglucose became growth limiting with only a slight decrease inexponential growth rate over the period.

At the end of this period of rapid growth, the pH control reagent waschanged to 6N potassium hydroxide solution and the glucose feed was keptconstant for the next 110 hours. During this period, ammonium, whichconcentration remained constant throughout exponential growth, wasdepleted from the medium and phosphate concentration dropped to lowlevels. Biomass continued to accumulate in the medium at a constantlinear rate despite the consumption of these key nutrients. Viable cellcounts also increased linearly until the ammonium was depleted from themedium, after which they remained constant. Ultimately, the fermentationproduced 71.5 g/L biomass dry weight.

COMPARATIVE EXAMPLE 1

Production Medium Components Concentration (g/L) Glucose 40.0 AmmoniumSulfate 8.0 Corn Steep Liquor 9.0 Potassium Phosphate, Monobasic 2.0Potassium Phosphate, Dibasic 1.0 Magnesium Sulfate 0.5 NaCl 0.5 CalciumChloride 0.1 Trace Metals: Boric Acid 0.00075 Cupric Sulfate 0.00006Potassium Iodide 0.00015 Ferric Chloride 0.0003 Manganese Sulfate 0.0006Sodium Molybdate 0.0003 Zinc Sulfate 0.0006 SAG 471 ® Antifoam 2 drops

The medium components of Comparative Example 1 were sterilized in asuitable manner to avoid any precipitation reactions and combined in asterile fermentor vessel. The complete, uninoculated medium was found tobe very dark with a strong odor characteristic of corn steep liquor.Candida tropicalis H5343 (ATCC 20962) was grown under sterile conditionson the medium listed in Table 4 in a stirred, aerated fermentor with aninitial liquid volume of 10 L. The sterile culture medium was inoculatedwith a 6% inoculum of Candida tropicalis H5343 (ATCC 20962) and grown at35° C. for approximately 9.5 hours with agitation and aeration ratessufficient to keep the dissolved oxygen above 20%. 5 ml of SAG 471antifoam was added to the fermentor during growth to control foaming inthe culture. The pH was maintained between 5.8-5.9 during the growthphase with the addition of 6N NH₄OH. When the culture stopped growingexponentially and the dissolved oxygen began to rise, the conversionphase was commenced by starting a continuous feed stream of Emersol®R267(a commercial grade of Oleic acid containing containing 71.8% oleicacid, 8.3% linoleic acid, 6.3% palmitic acid, 4.6% palmitoleic acid,2.8% stearic acid, 2.7% myristic acid, 0.93% C17:0 acid, 0.86%myristoleic acid, 0.58% linolenic acid, and 0.42% lauric acid) at a rateof 2.0 g/L/hr. Simultaneously, the temperature in the fermentor wasreduced from 35° C. to 30° C., the aeration rate was reduced to 1.2 vvm,and the pH control reagent was switched from NH₄OH to KOH. The pH wasmaintained at or above 5.8 during the conversion phase with 6N KOH. Acontinuous glucose feed stream was started to the fermentor at a rate of1.8 g/L/hr glucose at the end of the exponential growth immediatelypreceding the start of the conversion phase. This same glucose feed ratewas maintained to the fermentor throughout the conversion phase. 17 mlof SAG 471 antifoam was added to the fermentor during the first 3 hoursof the conversion phase to control very heavy foaming. After 50 hours ofthe conversion phase, the whole broth in the fermentor contained 64 g/Kgtotal dicarboxylic acids.

EXAMPLE 4 Modification of an Aqueous Suspension of Dodecanedioic acid

A 25 g/l finely-divided aqueous suspension of long-chain dicarboxylicacid was prepared by first, dissolving 12.5 grams of dodecanedioic acid,99%, a Dupont product (Aldrich Chemical Company) (DDDA), into waterusing excess KOH to form the water-soluble dicarboxylate. The solutionwas then rapidly titrated to pH 6 using H₂SO4 to precipitate the diacid.

Twenty milliliters of this suspension was distributed to each of eightflat bottom screw cap vials equipped with a stirring bar. Thus each vialcontained 0.5 grams of finely divided dodecanedioic acid suspended inwater. These were placed in a water bath at 30° C. on a magnetic stirrerand the suspension allowed to equilibrate with stirring overnight.

The effect of various additives on the suspension behavior was tested bybatch adding the test substances at various concentrations in theequilibrated vials. The compositions of the commercial test substancesare shown in Table 1. The net formulations in each of the vials areshown in Table 5.

TABLE 5 Additive Effects Testing for Aqueous Dicarboxylic AcidSuspensions Ratio of Amount Additive/DDDA Vial Additive (μg) (ppm) 1Oleic Acid 44 88 2 Oleic Acid 88 176 3 Methyl tallowate 44 88 4 Methyltallowate 88 176 5 Methyl oleate 44 88 6 Methyl oleate 88 176 7 None(control) 0 0 Test Volume = 20 ml Test Temperature = 30° C.Dodecanedioic acid in suspension = 0.5 grams

The appearance and behavior of the suspensions were observed 4 hours, 24hours, or longer after adding the test additives and compared with thecontrol (Vial 7). The control appeared as a suspension with someassociation of individual particles as viewed under a phase contrastmicroscope.

Both methyl ester additives at both concentrations gave betterassociation of the DDDA into discrete particles of similar size (Vials3-6). This was apparent when the vials were removed from stirring andallowed to settle. The suspensions with the methyl ester additivessettled much more rapidly than the control. The remaining aqueous phasewas clear compared to the control, which remained turbid with thesmaller DDDA particles remaining in suspension.

The most surprising results were obtained in suspensions with the oleicacid additives where in 4 hours, the particles began to associate intolarge masses. At twenty hours, Vial 1 had large dense masses of DDDAnear the bottom and some smaller masses at the top of the vial. Attwenty hours, Vial 2 contained a single sphere of DDDA about 1 cm indiameter and the aqueous phase was clear.

As vials 1 and 2 continued to be incubated beyond 20 hours, pieces ofthe larger masses had become broken. These pieces then attached to thestirring bar itself. Ultimately all the DDDA grew onto the stirring barsuch that the vial contained a whirling chunk of DDDA in clear water.

This example demonstrates how the addition of fatty acids and fatty acidderivatives may be used to make aqueous suspensions of dicarboxylicacids more amenable to separations by processes using filtration,sedimentation, or size classification.

EXAMPLE 5 Viscosity of Aqueous Dicarboxylic Acid Suspensions: Effect ofParticle Morphology

An approximately twelve-percent (117.0 g/kg) aqueous suspension ofsaturated and unsaturated dicarboxylic acids primarily C16-C18 wasprepared in a fermentation broth from high oleic acid sunflower oilusing a microorganism at 30° C. During the fermentation, methyl oleatewas added fed-batch to the fermentation broth at a rate to provide abouta 1% ratio of methyl oleate to dicarboxylic acids. A sample of broth wascollected for phase contrast microscopic observation and viscositymeasurements as shown in Table 6.

For comparison, the sample was then heated to 70° C. and rapidly cooledto room temperature to disrupt the disk structures that had formed inthe original broth. This was then subjected to similar analysis as isalso shown in Table 6.

TABLE 6 Effect of Particle Morphology on Brookfield Viscosity of anapproximately 12% Aqueous Fermentation Broth Suspension of DicarboxylicAcids. Particle Spindle Number/ Viscosity (cP) Sample Appearance SpindleSpeed (rpm) @ 21° C. Fermentation cup-shaped disks Spindle 2/ 5,000broth 25-30 μm diameter,   3 rpm 5 μm thick Fermentation needle-likecrystals Spindle 4/ 1,000,000 Broth Heated 0.3 rpm then cooled

A series of similar experiments were conducted at differentconcentration levels of dicarboxylic acid in suspension which confirmedthe above results since they behaved in substantially similar fashion.Such concentrations were as follows: 27.6 g/kg, 57.3 g/kg, 83.2 g/kg,95.8 g/kg, 100.9 g/kg, 117.0 g/kg, and 117.0 g/kg for a heat killedsample.

These results demonstrate the striking effect fatty acid derivativeshave on dicarboxylic acid morphology and viscosity in a fermentationbroth.

EXAMPLE 6 Effects of Various Fatty Acid Derivatives and Concentrationson Diacid Precipitate Morphology

Various levels and types of fatty acid derivatives were tested insimilar fermentations as in Example 5 and samples examinedmicroscopically to determine the effect on dicarboxylic acid particlegrowth. Table 7 shows several combinations of substrates used fordicarboxylic acid production, combinations of additive fatty acidderivatives, and resulting appearance under a phase contrast microscopeof dicarboxylic acids in suspension.

TABLE 7 Effect of Fatty Acid Derivative Additives on SuspensionMorphology in a Fermentation Broth Substrate being Oxidized toSuspension Modifiers Appearance of Suspended Dicarboxylic Acids Added(ratio to diacids) Dicarboxylic Particles High-Oleic Sunflower Fatty1.0% Methyl oleate Disk to cup-shaped, narrow Acids distribution around30 μm diameter by 5 μm thick High-Oleic Sunflower Fatty 0.1% Methyloleate Single narrow distribution disk- Acids shaped around 15 μmdiameter Partially hydrolyzed High-Oleic Contained 5% fatty acid Singlenarrow distribution disk- Sunflower Oil¹ glycerol esters shaped around15 μm diameter High-Oleic Sunflower Fatty None (comparative Bimodaldistribution of disk sizes Acids control for in situ fatty acidcentering around 5 and 10 μm oxidations) Hydrocarbons Mixture² 2.5%Emersol ® 267 + Disk-skaped particles 20 μm in 2.5% methyl tallowatediameter Hydrocarbons Mixture² 1.25% Methyl oleate + Single narrowdistribution disk- 1.25% Oleic Acid shaped around 15 μm diameterHydrocarbons Mixture² None (comparative Few disk shaped particles, mostcontrol for in situ particles < 2 μm hydrocarbon oxidations)¹high-pressure steam hydrolyzed oil having and Acid Value = 188.3 andSaponification Value = 198.5 indicating 95% hydrolysis of fatty acidtriglycerol esters leaving a mixture of mono-, di-, and tri-glyceridesto serve as precipitate modifiers. ²NORPAR ® 13 SOLVENT a product ofExxon Corporation containing normal aliphatic hydrocarbons C11 < 1%, C12= 13%, C13 = 50%, C14 = 35%, C15 < 1%.

EXAMPLE 7 Viscosity of Aqueous Dicarboxylic Acid Suspensions:Shear-Thinning Characteristics

The shear thinning characteristics of a 70.3 g/kg (˜7%) aqueoussuspension of C11-C15 saturated dicarboxylic acid was prepared in afermentation broth using 1.25% each methyl oleate and oleic acid. Phasecontrast microscopic examination of the broth showed the diacidsuspension to contain disk-shaped dicarboxylic acid particles 15 μm indiameter. FIG. 1 shows the shear thinning characteristics of thisaqueous suspension. A series of similar experiments were conducted atdifferent concentration levels of dicarboxylic acid in suspension whichconfirmed the above results since they behaved in substantially similarfashion. Such concentrations were 17.8 g/kg, 26.4 g/kg and 59.9 g/kg.

What is claimed is:
 1. A fermentation method which comprises conductingthe fermentation in a fermentation medium and modifying the rheologicalproperties of the fermentation medium containing a dicarboxylic acid byadding an effective dicarboxylic acid particle shape forming amount of afatty material to the fermentation medium to cause formation of shapeddicarboxylic acid particles, wherein the shape is selected from thegroup consisting of substantially disk shaped, substantially partiallyspherical shaped and substantially spherical shaped.
 2. A method formodifying the rheological properties of a fermentation medium containinga dicarboxylic acid according to claim 1 wherein the particles have adiameter greater than about 15 μm.
 3. A method for modifying therheological properties of a fermentation medium containing adicarboxylic acid according to claim 2 wherein the diameter of thedicarboxylic acid particles ranges from about 15 μm to about 1 cm.
 4. Amethod for modifying the rheological properties of a fermentation mediumcontaining a dicarboxylic acid according to claim 1 wherein thethickness of the substantially disk-shaped or substantially partiallyspherical shaped dicarboxylic acid particles ranges from about 1 μm toabout 15 μm.
 5. A method for modifying the rheological properties of afermentation medium containing a dicarboxylic acid according to claim 1further comprising maintaining the temperature of the fermentationmedium below about 50° C.
 6. A method for modifying the rheologicalproperties of a fermentation medium containing a dicarboxylic acidaccording to claim 1 wherein the fatty material is a fatty acid.
 7. Amethod for modifying the rheological properties of a fermentation mediumcontaining a dicarboxylic acid according to claim 1 wherein the fattymaterial is a fatty acid ester.
 8. A method for modifying therheological properties of a fermentation medium containing adicarboxylic acid according to claim 7 wherein the fatty acid ester is amethyl ester.
 9. A method for modifying the rheological properties of afermentation medium containing a dicarboxylic acid according to claim 1wherein the fatty material is added in an amount which provides greaterthan about 10 ppm based on the mass of the dicarboxylic acid.
 10. Amethod for modifying the rheological properties of a fermentation mediumcontaining a dicarboxylic acid according to claim 9 wherein the amountranges from about 10 ppm to about 5% based on the mass of thedicarboxylic acid.
 11. A method for modifying the rheological propertiesof a fermentation medium containing a dicarboxylic acid according toclaim 10 wherein the amount ranges from about 50 ppm to about 1% basedon the mass of dicarboxylic acid.
 12. A method for modifying therheological properties of a fermentation medium containing adicarboxylic acid according to claim 1 wherein the fatty material is acombination of fatty acid and fatty acid ester.
 13. A method formodifying the rheological properties of a fermentation medium containinga dicarboxylic acid according to claim 12 wherein the fatty acid esteris a methyl ester.
 14. A method for modifying the rheological propertiesof a fermentation medium containing a dicarboxylic acid according toclaim 1 wherein the fermentation medium includes (a) a source ofmetabolizable carbon and energy; (b) a source of inorganic nitrogen; (c)a source of phosphate; (d) at least one metal selected from the groupconsisting of an alkali metal, an alkaline earth metal, transitionmetals, and mixtures thereof; and (e) a source of biotin, substantiallyfree of particulate matter and bacteria.